Membrane protein function is regulated by the lipid composition of the bilayer in which the proteins are imbedded. This regulation has been described/explained using various terms: changes in bilayer fluidity; changes in bilayer compression or curvature frustration energy, which can be combined into a model of elastic bilayer deformations; changes in lateral pressure profile across the bilayer or in lipid packing stress; changes in bilayer free volume. Among these mechanisms, changes in bilayer can be ruled out, as they cannot explain changes in equilibrium distribution between different protein conformations. The remaining descriptions, though couched in different terms, can be regarded as different approaches to parametrize the lateral interactions among the bilayer-forming lipids and between the lipids and the imbedded proteins. The objective of the proposed research is to examine the energetic consequences of the hydrophobic coupling between membrane-spanning proteins and their host bilayer using an elastic bilayer model, which allows for rather intuitive quantification of the bilayer-protein coupling. In the simplest description, changes in bilayer elastic properties are evaluated in terms of the "pull" the bilayer imposes on an imbedded channel, which can be measured using gramicidin channels as force transducers. In more elaborate descriptions, the coupling can be expressed in terms of an experimentally determined spring constant, which in simple cases can be calculated a priori. The experiments will address the following questions. Can the elastic bilayer model be applied to multi-component lipid bilayers? This will be examined using bilayers composed of two phospholipids with different acyl chain lengths and headgroups, or two phospholipids plus cholesterol, where the spring constant will be determined by changing the hydrophobic mismatch between the bilayer and channel probe. Can the channel-bilayer hydrophobic mismatch "drive" a lateral association between bilayer-spanning channels? This will be studied by examining the relative stabilization of double-barreled channels of different lengths imbedded in bilayer of different thickness. Can pharmacological interventions modulate membrane protein function indirectly, by altering bilayer elastic properties? This will be examined by probing how selected drugs alter bilayer properties, and relating this information to more complex systems. How do changes in bilayer lipid composition alter complex channel function? This will be studied in parallel experiments on gramicidin channels and complex channels reconstituted into planar bilayers of varying composition.